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PubMed1984

Gel electrophoresis in studies of protein conformation and folding.

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.

Animals↗
PubMed2003

A chemometric approach to detection and characterization of multiple protein conformers in solution using electrospray ionization mass spectrometry.

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.

Algorithms↗
PubMed2004

Sampling protein conformations and pathways.

Protein flexibility and rigidity can be analyzed using constraint theory, which views proteins as 3D networks of constraints involving covalent bonds and also including hydrophobic interactions and hydrogen bonds. This article describes an algorithm, ROCK (Rigidity Optimized Conformational Kinetics), which generates new conformations for these complex networks with many interlocked rings while maintaining the constraints. These new conformations are tracked for the flexible regions of a protein, while leaving the rigid regions undisturbed. An application to HIV protease demonstrates how large the flap motion can be. The algorithm is also used to generate conformational pathways between two distinct protein conformations. As an example, directed trajectories between the closed and the occluded conformations of the protein dihydrofolate reductase are determined.

Algorithms↗
PubMed1997

Microsecond atomic force sensing of protein conformational dynamics: implications for the primary light-induced events in bacteriorhodopsin.

In this paper a new atomic force sensing technique is presented for dynamically probing conformational changes in proteins. The method is applied to the light-induced changes in the membrane-bound proton pump bacteriorhodopsin (bR). The microsecond time-resolution of the method, as presently implemented, covers many of the intermediates of the bR photocycle which is well characterized by spectroscopical methods. In addition to the native pigment, we have studied bR proteins substituted with chemically modified retinal chromophores. These synthetic chromophores were designed to restrict their ability to isomerize, while maintaining the basic characteristic of a large light-induced charge redistribution in the vertically excited Franck-Condon state. An analysis of the atomic force sensing signals lead us to conclude that protein conformational changes in bR can be initiated as a result of a light-triggered redistribution of electronic charge in the retinal chromophore, even when isomerization cannot take place. Although the coupling mechanism of such changes to the light-induced proton pump is still not established, our data question the current working hypothesis which attributes all primary events in retinal proteins to an initial trans<==>cis isomerization.

Bacteriorhodopsins↗
PubMed2004

Evaluation of the relative stability of liganded versus ligand-free protein conformations using Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method.

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space-filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log-likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site-directed mutagenesis in the context of protein engineering.

Amino Acids↗
PubMed1985

A model of protein conformational substates.

Many proteins have been observed to exist in a large number of conformations that are believed to play an important role in their dynamics. A model of protein conformational substates that incorporates the ideas of frustration and disorder in analogy to glasses and spin glasses is proposed. Applications to x-ray diffraction, Mössbauer studies, and recombination experiments are discussed.

Journal Article↗
PubMed2000

Biological effects of electromagnetic fields--mechanisms for the effects of pulsed microwave radiation on protein conformation.

Microwave exposure under "athermal" conditions occurs when no temperature rise can be measured by conventional thermometry. The existence of biological effects arising from the athermal exposure is still controversial, partly because of a lack of the linear dose response relation. We propose a model in which pulsed microwave radiation causes a triggering of the heat shock or stress response by altering the conformation of proteins through a transient heating of the protein and its close environment. We support this by modelling using the heat diffusion equation and show that pulsed exposure even when athermal can lead to transient temperature excursions outside the normal range. We propose that the power window phenomenon in which biological effects are observed at low power levels may be caused by an incomplete triggering of the heat shock response.

Animals↗
PubMed2005

Detection of coexisting protein conformations in capillary zone electrophoresis subsequent to transient contact with sodium dodecyl sulfate solutions.

Non-native conformations of proteins were generated by temporary contact with aqueous solutions of sodium dodecyl sulfate (SDS) and separated from the native state with capillary zone electrophoresis (CZE) in alkaline borate buffer deficient of SDS. Nine proteins at concentrations of 2.0 or 3.0 mg.L(-1) were compared in terms of their susceptibility to SDS. For superoxide dismutase and ferritin the tendency of unfolding was modest with < 25% of the protein being transformed to the non-native state at 10 mmol.L(-1) SDS. Highest susceptibility was observed for albumin, myoglobin (Mb), and hemoglobin with > 75% in the non-native state even at 2.0 mmol.L(-1) SDS. The influence of varying SDS concentrations on the conformational state of Mb was tested. Increasing the SDS concentration, circular dichroism revealed a reduction in alpha-helix, an increase in random coil, and an introduction of beta-sheet, which is absent in native structure. Modifications in the secondary structure were in agreement with distinct changes in the shape of the non-native Mb peak in CZE and make a gradual unfolding/refolding process with several coexisting molten globules instead of two-state transition of conformations most plausible for Mb. CZE was found to contribute to a further understanding of holo-Mb transformation towards a population of non-native conformations (i) by means of calculated peak area ratios of native to non-native states, which showed sigmoid transition, (ii) by detecting the release of the prosthetic heme group, and (iii) by changes in the effective electrophoretic mobility of the Mb-SDS peaks. Reconstituted holo-Mb forms differed in the Soret band around 410 nm, indicating diversity in the conformation of the heme pocket.

Carbonic Anhydrases↗
PubMed2002

Protein conformation and diagnostic tests: the prion protein.

BACKGROUND: Many clinical diagnostic tests depend on the accurate detection and quantification of proteins and peptides and their functions. Alterations of protein structure, and the resulting consequences on dynamics, can affect the outcome of laboratory tests. These changes can be a result of mutations, other in vivo factors, or even the experimental conditions of the diagnostic test. APPROACH: The relationship between protein structure and dynamics and experimentally observable properties used in diagnostic assays are discussed in light of transmissible spongiform encephalopathies, or prion diseases. CONTENT: This review describes current efforts and possible future directions of prion diagnostic development. Recent advances in therapeutic development are also addressed. SUMMARY: The intent of the review is to highlight the role of protein dynamics and conformational change in protein-based diagnostics and treatments for prion disease.

Animals↗
PubMed1990

The conformation-sensitive immunoassay: a membrane based ELISA system for identifying antibodies sensitive to alterations of protein conformation.

A Conformation-Sensitive Immunoassay (CSI) has been developed for identification of antibodies that are sensitive to alterations in protein conformation. The method involves covalently coupling proteins to an activated hydrophilic membrane support. The membrane bound proteins are then treated under conditions known to alter protein conformation, immobilized in a non-native state via additional covalent interactions with the support, and subsequently probed using conventional ELISA techniques. This method has been validated using several well characterized conformation-sensitive antibodies to horse cytochrome c and sperm whale myoglobin. A panel of monoclonal antibodies directed against bovine somatotropin (bSt) has been partially characterized using this validated CSI procedure. Each of these antibodies to bSt has been shown to detect conformational alterations of bSt structure. Data are also presented that demonstrates that the accuracy of CSI analysis is superior to that of Western blotting for characterizing conformation-sensitive antibodies.

Animals↗
PubMedDate not supplied

Statistical mechanical treatment of protein conformation. I. Conformational properties of amino acids in proteins.

A statistical mechanical (one-dimensional Ising model) treatment, based on the dominance of short-range interactions, is developed in this series of papers; it is intended as an improvement over empirical prediction schemes for obtaining approximate initial conformations of proteins (to be used to try to deduce the native conformation by subsequent energy minimization). In the present paper, the statistical weights for a two-state model (alpha-helical and other conformations) and for a three-state model (alpha-helical, extended, and other conformations) are evaluated from x-ray data on 16 native proteins. The method for evaluating the statistical weights is presented. Asymmetric alpha-helical nucleation parameters are also evaluated for the 20 naturally occurring amino acids. On the basis of these statistical weights, the conformational properties of the twenty naturally occurring amino acids are discussed. The statistical weights evaluated from x-ray data are also discussed in comparison with experimental results on the helix--coil transition in polyamino acids in solution. The predominant role of short-range interactions, and some possible long-range effects in determining the statistical weights, are discussed in conjunction with the mechanism of protein folding.

Amino Acids↗
PubMed1998

Application of automated methods for determination of protein conformational stability.

Automation of protein stability determination saves much time and often results in better data. In our laboratory we estimate that the lack of such instrumentation would increase the amount of time required to make and characterize a mutant protein by at least one-third. As with any experiment, lack of attention to important details can required repeating the experiment or, worse, give superficially good data that are flawed in some manner. Nevertheless, with a minimal investment of attention, an automated instrument can give dramatic gains in productivity.

Enzyme Stability↗
PubMed2002

Potential involvement of copper and thiol-disulphide interchange in prion proteins' conformational conversion.

The prion protein PrP(C) in transmissible spongiform encephalopathy converts to the pathogenic isoform PrP(Sc) containing less alpha-helical structure and a greater beta-pleated sheet content. The stability of PrP(C) protein is partly dependent on the disulphide bond between two alpha-helices designated B and C. Further stability could arise from ligand complexes of Cu(II) ions formed with carboxylic acid side chains in PrP(C). Electron spin resonance (E.S.R.) spectra and atomic absorption measurements have shown for alpha-keratin that the formation of ligands by Cu(II) is 10(2) more rapid than interaction of Cu(II) with ionised thiols X-S(-) which form X-S-Cu(+). X-S(-) destabilises disulphide bonds by thiol-disulphide interchange. When insufficient Cu(II) is present to form ligands with all available sites in PrP(C) then unblocked X-S(-) groups could potentially destabilise the disulphide bonds by thiol-disulphide interchange followed by reformation of the disulphide bond in the beta form of PrP(Sc) and the release of X-S(-) to interact with other PrP(C).

Animals↗
PubMed1996

Protein conformation and disease.

In a number of neurodegenerative diseases, a change in the conformation of specific proteins occurs which is considered to be related to the development of the disease process. In general, a change in the protein conformation from a predominantly alpha-helical to a predominantly beta-sheet structure, due to genetic or environmental factors, leads to oligomerization of the proteins. In Alzheimer's and prion diseases, insoluble precipitates or plaques are often observed in vivo as a consequence of beta-fibril formation due to the polymerization of the proteins. In certain other neurodegenerative diseases, for example in Huntington disease, the beta-sheet oligomers may interact differently with other functional proteins compared to the monomeric proteins inducing the disease. Biophysical studies would provide information regarding the forces responsible for the oligomerization of these proteins and hence provide methods for detecting these diseases, leading eventually to therapeutic approaches.

Alzheimer Disease↗